Abstract
Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward Pfr → Po transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse Po → Pfr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm−1. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.
Highlights
Phytochromes, bacteriophytochromes, and cyanobacteriochromes (CBCRs) are among a superfamily of linear tetrapyrrole binding photoswitching proteins [1,2,3]
In line with our previous study [15], a home-built LED box that fits four 5 mm throughIn line with our previous study [15], a home-built LED box that fits four 5 mm hole LEDs was implemented to keep the samples in their respective conformers for all through-hole LEDs was implemented to keep the samples in their respective conformers the spectroscopic experiments
To achieve conversion toward the 15EPo conformer of Bbv, a tungsten lamp with a 650 nm longpass filter was used since our available LEDs did not convert the CBCR sample fast longpass filtersample was used sincewas ourthen available
Summary
Phytochromes, bacteriophytochromes, and cyanobacteriochromes (CBCRs) are among a superfamily of linear tetrapyrrole (bilin chromophore that is metabolically derived from heme) binding photoswitching proteins [1,2,3]. In nature, these proteins act as photosensing and signaling units that allow organisms to detect and respond to light from the UV to near-IR range [4,5,6,7,8]. CBCRs have shown to be a versatile tool in optogenetic applications by requiring only a single compact domain for incorporation of the cofactor chromophore, the photosensing unit that can be considered as the active site [3,14]. We uncovered the initial ultrafast dynamics as the phycocyanobilin (PCB cofactor in protein matrix) photoswitches from the Pg to Pr and Pr to Pg conformers in a reversible manner (controlled by a combination of actinic laser pump pulses and continuous-wave/cw LEDs)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
